We have studied the regulation of muscarinic receptor and delta-opioid receptors in two related neurohybrid cell lines, NCB-20 and NG 108-15 cells. Our previous studies have demonstrated that treatments of NCB-20 cells with butyrate and dibutyryl cAMP result in up- and down-regulation in the number of muscarinic acetylcholine receptors (mAChRs), respectively. Our current results show that NCB-20 cells express the mRNA for m1-and m4-mAChRs. Butyrate and dibutyryl cAMP induce a respective increase and decrease in the mRNA levels of m1-and m4-mAChRs. Moreover, ethanol treatment, which has been shown to up-regulate mAChRs, also increases the levels of m1-and m4-mAChR mRNA. mAChR binding sites can be paradoxically up-regulated by stimulation with certain mAChR agonists, such as pilocarpine and oxotremorine, but are down-regulated by other agonists, such as carbachol, oxotremorine-M and McN-A343. Additionally, we have further characterized delta-opioid binding sites associated with the nuclear matrix of NG108-15 cells. The binding sites of the delta-specific antagonist 3H-naltrindole and the mixed agonist- -antagonist 3H-diprenorphine are enriched in nuclear matrix preparations. However, agonists bind with low affinity, if at all, to nuclear matrix preparations. Adenylate cyclase activity is not detected in this preparation; however, opioid inhibition of adenylate cyclase activity was found in the nuclear preparation. Colchicine treatment causes up-regulation in the plasma membrane but downregulation in the nuclear matrix sites. Protein kinase A treatment or etorphine prestimulation of cells abolishes the antagonist binding in the nuclear matrix but not in the plasma membrane or nuclear membranes. However, a decrease in the agonist binding affinity and density was found in the plasma membrane. Our results suggest that nuclear membrane delta-opioid receptors represent newly synthesized molecules enroute to the cell surface, whereas the nuclear matrix contains a subpopulation of internalized delta-sites.